Apparatus and method for concurrent chemical synthesis

ABSTRACT

This invention provides an apparatus for preparing chemical libraries. The apparatus includes (1) a carousel comprising a plurality of reaction mounts having at least one reaction well; (2) a rotator that rotates the carousel step-wise; (3) a fluid delivery system; (4) a drain system; and (5) a programmable computer that controls the operation of the apparatus, including the rotator, the fluid delivery system, the drain system and other systems in the apparatus. The preparation-of chemical libraries involves rotating the carousel through a plurality of stations. At each station, a physical step in a reaction protocol is carried out on the reaction wells of the mount docked at the station.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of the priority date of U.S.Provisional Patent application 60/113,571, filed Dec. 22, 1998.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] This invention is directed to the fields of mechanical devicesand methods of organic chemistry. More particularly, this inventionprovides a machine for preparing chemical libraries and methods ofpreparing those libraries.

[0004] Chemical libraries are collections of different chemicalcompounds, usually of the same class. Chemical libraries are useful inscreening methods to determine whether any of the compounds haveparticular properties. Libraries of nucleic acids are particularlyuseful in hybridization analysis to detect the presence of targetnucleic acids in a sample.

[0005] Methods in combinatorial chemistry are useful in the creation ofchemical libraries. The methods usually involve adding different unitssequentially to a base molecule, either randomly or by design.Apparatuses have been described that produce libraries of nucleic acids.These include, for example, U.K. Patent 2,194,176 (Nicholson), U.S. Pat.No. 5,288,468 (Church et al.), U.S. Pat. No. 5,445,934 (Fodor et al.)and U.S. Pat. No. 5,472,672 (Brennan). Improved apparatuses thatincrease the speed at which libraries of molecules can be made would bea useful addition to the art.

SUMMARY OF THE INVENTION

[0006] This invention provides an apparatus for preparing chemicallibraries in which the members of the library are prepared in parallel,continuous reactions, rather than batch-wise. This arrangement allowsmore rapid synthesis of the libraries. The apparatus is especiallyadapted to the preparation of libraries of polymers, such as nucleicacids, polypeptides and peptide nucleic acids. The apparatus includes arotatable carousel that contains a plurality of reaction mounts. Eachreaction mount comprises at least one reaction well arranged on a radiuswith respect to the axis. The radii are spaced apart at equal angles sothat the wells are arranged in at least one concentric circle around theaxis. The apparatus also contains a rotator that rotates the carouselstep-wise around the axis. Each incremental step docks each of thereaction mounts at a different reaction station where a physical step inthe chemical protocol takes place. The reactions steps generally involveat least fluid delivery to a reaction well, drainage of fluid from awell, and incubation or wait (null) steps, in which fluid is neitheradded to nor removed from a well. Accordingly, the apparatus of thisinvention includes a fluid delivery system that delivers liquid to atleast one reaction well in each of a plurality of docked reaction mountsand a drain system that drains liquid by differential pressure from atleast one reaction well of each of a plurality of docked reactionmounts. The rotation of the carousel and the physical steps arecontrolled by a programmable digital computer.

[0007] The creation of chemical libraries involves the creation ofchemical linkages in a parent molecule. Usually the process isiterative, generating multiple new linkages. Frequently, as is the casefor polymers, the generation of a linkage involves coupling a componentto the parent molecule. The creation of a chemical linkage, in turn, canbe divided into a number of sequential chemical steps. For example, thecreation of a phosphodiester bond in the synthesis of a nucleic acidgenerally involves deprotecting a sugar moiety of a parent molecule,coupling a reactive phosphoramidite to the sugar moiety, oxidizing thephosphoramidate bond into a phosphotriester bond, and capping unreactedmolecules. Ultimate removal of a protecting group generates thephosphodiester. This procedure can, in turn, be broken down into thephysical steps of adding liquid containing reagents or wash solutions toa reaction well containing the parent molecule, removing liquid from thewell and incubating, or waiting. These physical steps are carried out atthe stations designated by the apparatus. The stations are arranged in acircle around the carousel. Each station performs, in sequence, one ofthe physical steps of the reaction. Thus, as a reaction mount moves fromstation to station the set of sequential steps is performed on the wellsof the reaction mount so that after one complete rotation, the entireseries of steps has been performed on the reaction wells of the mount.In this way, the chemical linkage is established on the parent moleculein the well. The parent molecule is reversibly immobilized on aninserted solid support, such as glass or an inert polymer or plasticsuch as polystyrene, polyethylene or Teflon™ (available from, e.g., PEBiosystems). However, the reaction mounts also are arranged in a circlearound the carousel. Therefore, at each incremental step, a differentphysical step in the process is carried out on the wells of one of thereaction mounts. Consequently, the creation of the linkages is carriedout in parallel, with each reaction mount at a different stage of thereaction. Thus, the methods of this invention do not require all thereaction wells to pass through a single step in the reaction before anyother reaction well can continue on the next step. This savesconsiderable time in the process.

[0008] The creation of a library is limited by the number of reactionwells that one can perform a physical step on at any one time. Forexample, if one wishes to prepare 192 different compounds, and one hasthe capacity to perform a physical step on eight reaction wells at apass, it would require twenty-four passes before the next step canbegin. In contrast, by performing the steps in parallel on eight wellsat a time, twenty-four sets of wells are actively engaged in thechemical steps.

[0009] In one aspect this invention provides an apparatus comprising: a)a carousel that is rotatable around an axis, the carousel comprising aplurality of reaction mounts, each reaction mount comprising at leastone reaction well arranged on a radius with respect to the axis, theradii spaced apart at equal angles, whereby the wells are arranged in atleast one concentric circle around the axis; b) a rotator that rotatesthe carousel step-wise around the axis, each incremental step dockingeach of the reaction mounts at a separate station; c) a fluid deliverysystem that delivers liquid to at least one reaction well in each of aplurality of docked reaction mounts; d) a drain system that drainsliquid by differential pressure from at least one reaction well of eachof a plurality of docked reaction mounts; and e) a programmable digitalcomputer that controls the rotator, the fluid delivery system and thedrain system.

[0010] In one embodiment of the apparatus: (i) each reaction wellcomprises a drainage hole; (ii) the carousel comprises a plate whichcomprises a plurality of liquid conduits that connect with the drainageholes and are engagable with the drain system; and (iii) the drainsystem is a vacuum drain system comprising: (1) a plurality of vacuumlines that connect with vacuum source and (2) conduit engagement meansthat engage the vacuum lines with a plurality of the liquid conduitswhen the reaction mounts are docked at a station, whereby liquid in thereaction wells is drained through the vacuum lines.

[0011] In a further embodiment of the apparatus: (i) each liquid conduitcomprises: (1) a depression in the plate below the reaction mount whichforms a chamber with the reaction mount, wherein the chambercommunicates with the drainage holes of the reaction mount; (2) an exitport exiting under the plate; and (3) a bore through the plate theconnects the chamber with the exit port; and (ii) the conduit engagementmeans comprises: (1) a non-rotating connector plate positioned under thecarousel; the connector plate having an engagement port that isengagable with the exit port positioned at each station, wherein each ofa plurality of the engagement ports is connected to a vacuum line; and(2) an actuator that raises the connector plate to engage the pluralityof engagement ports with the plurality of exit ports.

[0012] In a further embodiment of the apparatus the fluid deliverysystem comprises: (i) an assembly positioned above the carousel, theassembly comprising a plurality of dispensing modules mounted at each ofa plurality of the stations, each dispensing module comprising adispensing head adapted to deliver fluid to the well of a reaction mountdocked at the station; (ii) a plurality of fluid dispensers, eachdispenser adapted to dispense an amount of fluid; (iii) a plurality offluid lines, each fluid line connecting a fluid dispenser to adispensing head.

[0013] In another aspect this invention provides a method for performingin parallel a series of physical steps in a chemical reaction protocol,wherein the protocol generates a chemical linkage in a parent molecule.The method comprises: a) providing a carousel that is rotatable aroundan axis, the carousel comprising a plurality of reaction mounts, eachreaction mount comprising at least one reaction well arranged on aradius with respect to the axis, the radii spaced apart at equal angles,whereby the wells are arranged in at least one concentric circle aroundthe axis, wherein each well comprises the parent molecule attached to asolid support; b) rotating the carousel step-wise around the axis atleast once, each incremental step docking each of the reaction mounts ata separate station, wherein (1) each station is dedicated to perform aphysical step in the series during a docking, wherein the physical stepsinclude adding a liquid to a well, draining a liquid from a well, andincubating; and (2) the stations are arranged to perform the series ofphysical steps in sequence; and c) performing, with each rotation of thecarousel, the series of physical steps in a reaction well of each of atleast two of the reaction mounts, whereby a chemical linkage isgenerated in the parent molecule.

[0014] In another aspect this invention provides a method for performingin parallel a series of physical steps in a chemical protocol. Themethod comprising the steps of: a) providing a carousel that isrotatable around an axis, the carousel comprising a plurality ofreaction mounts, each reaction mount comprising at least one reactionwell arranged on a radius with respect to the axis, the radii spacedapart at equal angles, whereby the wells are arranged in at least oneconcentric circle around the axis, wherein each well comprises theparent molecule attached to a solid support; b) providing a rotator thatrotate the carousel step-wise around the axis, each incremental stepdocking the reaction mounts a station, wherein: (1) each station isdedicated to perform a physical step in the series during a docking and(2) the stations are arranged in series from an initial station thatperforms an initial physical step in a series of physical steps in achemical protocol to a final station that performs a final physical stepin the series of physical steps; c) performing an initial rotation ofthe carousel around the axis, wherein the stations begin to perform theseries of physical steps as a reaction mount docks at the initialstation; and d) performing a final rotation of the carousel around theaxis, wherein the stations cease to perform the series of physical stepsas a reaction mount docks at the final station. The initial and finalrotations result in one complete series of steps on a reaction well ofeach reaction mount.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 depicts the apparatus of this invention.

[0016]FIG. 2 depicts a reaction mount.

[0017]FIG. 3 depicts a carousel and liquid conduit.

[0018]FIG. 4 depicts a drain system.

[0019]FIG. 5 depicts a fluid delivery system.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0020] I. DEFINITIONS

[0021] Unless defined otherwise, all technical and scientific terms usedherein have the meaning commonly understood by a person skilled in theart to which this invention belongs. The following references provideone of skill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

[0022] “Parent molecule” refers to a molecule bound to a solid support,including a linking group through which a first component issubsequently bound to the solid support.

[0023] “Component” refers to an atom or molecule linked to a parentmolecule in a chemical reaction. In one embodiment, the component is amonomer.

[0024] “Monomer” refers to a chemical compound that can be chemicallylinked in iterative fashion in the generation of a polymer.

[0025] “Polymer” refers to a compound comprising a series of monomersconnected through a chemical linkage. Polymers include, withoutlimitation, nucleic acids (nucleotides joined by phosphodiesterlinkages), polypeptides (amino acids joined by amide linkages),polysaccharides (monosaccharides joined by glycosidic linkages), variousnucleic acid analogs (e.g., peptide nucleic acids), polyurethanes,polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,polyarylene sulfides, polysiloxanes, polyimides and polyacetates.

[0026] “Scaffold molecule” refers to a parent molecule which is not apolymer. Scaffold molecules can be substituted at various positions byone or more chemical linkages with various components. Scaffoldmolecules also can be modified in existing chemical linkages by, e.g.,oxidation or reduction. Molecules bearing a common skeleton that canfunction as scaffold molecules include diazepines and other smallmolecules, such as described in U.S. Pat. No. 5,288,514 (Ellman),hydroxystillbenes, urea linked diamines, phosphonic acid esters,beta-turn mimetics, pyrazoles, isoxazoles, miconazole analogs,aminoproline analogs, piperazinediones, hydantoins using a carbamatelinker, 1-phenyl-pyrazolones, imidazoles, beta-lactams, pyrrolidines,quinolones, thiazolidines, pyridines, pyridopyryimidines, carbolines,hetrocyles, piperazines, polyazacyclophanes, quinolines and tertiaryamines. (See, e.g., THE COMBINATORIAL INDEX.)

[0027] In the methods of this invention each rotation of the carouselresults in one complete reaction cycle that creates a new linkage.Additional cycles create further linkages. In the case of a polymer,iteration of the process results in the addition of new moieties to theend of a growing chain. In the case of a scaffold compound, theiterative process can mean reacting a different site on the compound tocreate a new linkage.

[0028] “Nucleic acid” refers to a polymer composed of nucleotide units(ribonucleotides, deoxyribonucleotides, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof) linked via phosphodiester bonds, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof. Thus, the term includes nucleotide polymers in which thenucleotides and the linkages between them include non-naturallyoccurring synthetic analogs, such as, for example and withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2′-O-methyl ribonucleotides, peptide-nucleicacids (PNAs), and the like. Such polynucleotides can be synthesized, forexample, using an automated DNA synthesizer. The term “oligonucleotide”typically refers to short polynucleotides, generally no greater thanabout 100 nucleotides. It will be understood that when a nucleotidesequence is represented by a DNA sequence (i.e., A, T, G, C), this alsoincludes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

[0029] “Polypeptide” refers to a polymer composed of amino acidresidues, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. The term “peptide” typicallyrefers to short polypeptides containing from 2 to about 50 amino acids.The term “protein” typically refers to large polypeptides containingmore than about 50 amino acids.

[0030] “Chemical library” refers to a collection of compounds ofdifferent structures. Generally, the compounds will fall into the sameclass of chemical compounds, e.g., DNA, polypeptides, benzodiazepines,etc. Libraries of compounds can be divided into two main classes. Afirst class is libraries of polymers. A second class is libraries offunctionalized scaffold molecules. In either case, the various chemicallinkages that can be created with the methods of this invention are atthe discretion of the practitioner.

[0031] “Chemical linkage” refers to one or more chemical bonds having aparticular chemical character. These include, without limitation, aphosphodiester bond, a phosphorothioate bond, a phosphonate bond, aphosphoramidate bond, an amide bond, an imine bond, a carbamate bond, anazo bond, a sulfone bond, a sulfonide bond, a sulfonamide bond, asulfide bond, a disulfide bond, an ether bond, an ester bond, a thioureabond, a urea bond and a carbon-carbon bond (single, double or triplebond).

[0032] “Plurality” means two or more.

[0033] II. APPARATUS FOR CONCURRENT CHEMICAL SYNTHESIS

[0034] A. OPERATING PARTS

[0035] Referring to FIG. 1, the apparatus of this invention includes (1)a carousel 100 comprising a plurality of reaction mounts 105 having atleast one reaction well 106; (2) rotator 120 that rotates the carouselstep-wise; (3) a fluid delivery system 130 and 135; (4) a drain system140; and (5) a programmable computer 150 that controls the operation ofthe apparatus, including the rotator, the fluid delivery system, thedrain system and other systems in the apparatus. Optionally, theapparatus can include a temperature controlling system 160 forregulating temperature of a reaction mount docked at a station and anoptical analyzing system 170 for analyzing fluid in a well of a reactionmount docked at a station. In another embodiment, the apparatuscomprises a containment system 180 that separates the carousel, drainand fluid delivery systems from the exogenous environment and allows thechemical reaction to occur in an environment of choice (e.g., N₂).

[0036] B. CAROUSEL

[0037] The apparatus comprises a carousel that comprises a plurality ofreaction mounts having reaction wells, in which the protocol isperformed. In a preferred embodiment, the carousel comprises a circularplate 100 on which reaction mounts 105 can be mounted. In oneembodiment, the plate is about 0.475 inches thick, 12 inches in diameterand is made of anodized aluminum. However, the carousel could take otherconfigurations and materials, e.g., a hub with spokes that support thereaction mounts. The number of reaction mounts held by the carousel isnot critical. However, it is very efficient to include as least as manymounts as there are physical steps in the chemical protocol. In apreferred embodiment of this invention, the carousel holds 24 reactionmounts, and the protocol includes 24 steps.

[0038] The reaction mounts generally are removably insertable onto thecarousel. They can be composed of any material that resists reactionwith the chemicals that are placed in the reaction wells. For example,stainless steel is an attractive material. Various plastics, such aspolyethylene or polypropylene also are useful. In a preferred embodimentthe mounts are approximately ¼-inch thick anodized and teflonizedaluminum solid rectangular polygons of approximately 0.3-inch wide and3.8 inches in length. These mounts will be fixed statically to thecircular plate by screws at either end, positioned radially from adistance form the center of 2.5 inches out to the edge of the plate.

[0039] Referring to FIG. 2, reaction mount 200 includes at least onereaction well 210. The function of the reaction wells is to provide avessel to hold a solid support 220 on which a parent molecule will beattached and to provide a volume for carrying out the reactions. In apreferred embodiment, the reaction mounts contain a plurality ofreaction wells, e.g., eight wells. In general, the wells are arranged soas to be accessible to the fluid delivery and drain systems. In apreferred embodiment, the wells are arranged linearly. The line of wellsis positioned along a radius of the plate, so that at each station, thefluid handling devices can work along the radius. In a preferredembodiment, each well comprises a drain hole 230 in its bottom, throughwhich liquid can drain out of the well.

[0040] In certain embodiments, the reaction mounts of this invention canbe removed from the carousel and assembled into “microtiter plates.”Microtiter plates typically have a multiple of 96 wells arranged in rowsand columns. For example, a 96-well plate could have 8 rows and 12columns. A 384-well plate could have 16 rows and 12 columns.Accordingly, the number of wells on a reaction mount can be chosen tomeet this arrangement, e.g., 8 wells, 12 wells, 16 wells, etc. In oneembodiment, the wells of a reaction mount are configured as those in amicrotiter plate, about 9 mm apart center-to-center and about {fraction(7/32)}″ in diameter.

[0041] The wells are adapted to contain solid supports 220 on which theparent molecule is attached. The solid supports can be made of any inertmaterial that will not react with the fluids added to the well. Inertplastics or glass are preferred. Also, it is useful to attach the parentmolecule to the solid support by means of a cleavable linkage, such assuccinate linker, in order to isolate the molecules from the support.

[0042] In a preferred embodiment the carousel also functions to transmitliquid from the reaction wells to the drain system. The carouseltransmits liquid through a series of conduits that connect holes in thereaction well with the drain system, when the drain system is engagedwith the plate. Preferably, a single bore in the plate connects thedrain system with all the holes in a reaction mount. Referring to FIG.3, the carousel plate 300 comprises a depression 310 that runshorizontally the length of the mount. The bottom of reaction mount 320covers this depression, forming a chamber 330. O-ring 340 on the bottomof the mount seals this chamber. The screws holding the mount to theplate will provide the normal force to form the seal. On the bottom ofeach depression is a single bore 350 through the plate. The depressionitself is sloped towards the drain bore to include gravity in the forcesdraining the chamber. This bore through the disc terminates in exit port360, e.g., a Teflon™ or stainless steel nipple, that serves as theconnection for a vacuum drain system. Thus, liquid can be drained from awell through a hole in the well, collected into the depression in theplate and flow out through the bore in the plate.

[0043] C. ROTATOR

[0044] One aspect of this invention is rotating the reaction mountsthrough a number of stations where a step in the reaction protocol takesplaces. The invention provides for this purpose a rotator that rotatethe carousel in a step-wise fashion through a number of incrementalsteps. Each step rotates the carousel a designated number of equaldegrees. Thus, the rotator stops the reaction mounts at each of a numberof stations defined by the stopping positions. At each station, theapparatus performs one of the physical steps in the protocol. The numberof stations is selected to accommodate the number of physical steps inthe protocol and the reaction mounts loaded on the carousel.

[0045] In general, the number of degrees for each step will equal 360°divided by the number of stations. For example, in one embodiment, amotor rotates the carousel through twenty-four stations in 15° steps.Alternatively, there could be 18 stations at 20° intervals, 36 stationsat 10° intervals, etc.

[0046] In order to have each reaction mount stop at a station with eachstep, the reaction mounts must be placed around the carousel atintervals equaling a multiple of the angle between stations. Forexample, if the stations are set 15° apart, the reaction mounts can bestationed at 15° intervals. Alternatively, fewer reaction mounts can beplaced at multiples of 15° (e.g., 12 mounts at 30° or 8 mounts at 45°)or combinations thereof, so that with each incremental step, a reactionmount stops at a station. In such cases, each step will move a mount toa station, but certain stations may be left unoccupied and will bedormant during that step.

[0047] The rotator is any rotary activator to effect a mechanicalrotation including, e.g., an electromechanical motor, a pneumaticactivator or hydraulic activator that functions as a microsteppingstepper motor. The motor can engage the carousel through an axle.However, the motor can be connected to the carousel by other means, suchas a belt or lever. In one embodiment, the motor is positioned above theplate and rotates the plate by means of an axle connecting the motor tothe plate. Position feedback for the motor can be provided by an opticalencoder and by IR sensor for homing.

[0048] At each station one physical step in the protocol is performed onthe reaction wells of a reaction mount docked there. These steps includeadding liquid to a well, draining liquid from a well and incubation or anull step. As discussed below, other steps are contemplated includingincubating with heating or analysis of the materials in the well. In anycase, the apparatus is adapted so that devices for fluid delivery, fluiddrainage or other functions are positioned to work at the stationsdefined by the steps of the motor.

[0049] D. DRAIN SYSTEM

[0050] The use of the apparatus involves draining liquid deposited in areaction well, with or without simultaneous adding of liquid to the well(e.g., washing). Accordingly, the apparatus of this invention includes adrain system that removes liquid from the reaction wells. The drainsystem can drain liquid from the wells by any means. In a preferredembodiment, the apparatus is adapted to drain liquid from a hole in thebottom of the wells. This can be accomplished by gravity, butpreferably, drainage is active, employing a pressure differential. Thisincludes, preferably, a vacuum to suck liquid from the well, orover-pressure applied to the top of a well to push liquid out. Inanother embodiment, drainage is achieved by sucking liquid (e.g., by atube) from the top opening of the well, to which liquid is added.

[0051] In a preferred embodiment, the drain system is a vacuum drainsystem that removes liquid from a hole in the bottom of a well bysuction. In this embodiment, each reaction well includes a drainage holein the bottom through which liquid can drain. The carousel platecomprises a plurality of conduits terminating, e.g., in an exit port,that connects the drainage holes to the drain system when the drainsystem is engaged with the carousel. More specifically, referring backto FIG. 3, each liquid conduit comprises (1) depression 310 in the platebelow reaction mount 320 which forms chamber 330 with the reactionmount, wherein the chamber communicates with drainage holes 325 of thereaction mount; (2) exit port 360 exiting under the plate; and (3) bore350 through the plate that connects the chamber with the exit port.

[0052] Referring to FIG. 4, the drain system can comprise conduitengagement means to engage the conduits and drain liquid from them. Theconduit engagement means can comprise (1) non-rotating connector plate400 positioned under carousel 405. The connector plate has an engagementport 410 that is engagable with the exit port 420 positioned at eachstation, wherein each of a plurality of the engagement ports isconnected to a vacuum line 430; and (2) an actuator 440, e.g., apneumatic cylinder, solenoid or linear motor, that raises the connectorplate to engage the plurality of engagement ports with the plurality ofexit ports. The exit port and the conduit engagement means preferablyare complementary mechanical parts that fit each other. For example, theexit port can comprise a nipple (a hollowed protrusion) that fits withnozzles of the engagement means. This arrangement can be reversed.

[0053] The conduit engagement means, e.g., nozzles, are arranged aroundthe connector plate at locations that correspond to the stations atwhich the reaction mounts will stop. For example, the nozzles can bearranged circularly at equal intervals (e.g., 15° intervals for a devicewith 24 stations) and at a radius placing them just below the exitports. At each incremental step, the drain system is engaged with thecarousel by raising the connector plate to engage the engagement meanswith the exit ports. This connection is airtight. Vacuum lines areattached to the nozzles that are at locations designated as drainstations. The other nozzles are not connected to vacuum lines, thus nodraining takes places at those stations. The application of vacuum sucksthe liquid from the wells. Then the connector plate is lowered to allowrotation of the carousel in the next step.

[0054] Vacuum lines 430 connect through manifold 450 with vacuum source460, e.g., a vacuum pump or any source of vacuum that can suck liquidfrom the vacuum lines, and conduit engagement port 410 that engage thevacuum lines with a plurality of the liquid conduits when the reactionmounts are docked at a station. In this way, liquid in the reactionwells is drained through the vacuum lines and into a trap. From manifold450, a tube 470 runs through a valve and to a vacuum trap 480. The trapis evacuated using vacuum pump 490 that can withstand the corrosivechemicals used in synthesis. After the cylinder raises the plate, thevacuum drain is activated by opening the valve to the trap. At the endof the drain time, this valves is closed and the plate is lowered (Thiscan occur while the dispensing is taking place.) When the valve is open,a vacuum is created in the manifold, in the vacuum tubes, and in thedepressions below the reaction mounts located at drain stations. Thisserves to drain the wells in those stations.

[0055] This arrangement allows great flexibility as drain stations canbe moved, added, or deleted simply by connecting or disconnecting a tubefrom the station nozzle to the manifold.

[0056] E. FLUID DELIVERY SYSTEM

[0057] The apparatus also includes a fluid delivery system that deliversfluid to the wells of reaction mounts docked at stations designated forfluid delivery. Referring to FIG. 5, the fluid delivery system includes,in one embodiment, assembly 500 positioned above carousel 510. Theassembly includes a plurality of dispensing modules 520 mounted at eachof a plurality of the stations. Each dispensing module comprises adispensing head 530 adapted to deliver fluid to the well of a reactionmount docked at the station. The fluid dispensing system also includes aplurality of fluid dispensers 540, each dispenser adapted to dispense anamount of fluid. The system also includes a plurality of fluid lines550, each fluid line connecting a fluid dispenser to a dispensing head.

[0058] The dispensing modules can deliver liquid with positivedisplacement pumps. The dispensing head can include tube ends that arelocated above each well. Alternatively, the dispensing heads can belocated above the reaction mounts and moved by, e.g., stepper motors andlead screws along the length of the reaction mounts. The motion relativeto the carousel is radial (e.g., from outer diameter to inner diameter).The dispensing head stops above the center of each well in the reactionmount. If the synthesis program determines that fluid is to be added tothat particular well, then the syringe pump delivers the designatedvolume.

[0059] For some dispensing stations, e.g., wash stations, there is asingle tube end. But for there are some processes that either requiremultiple fluids to be dispensed concurrently or a single fluid from achoice of, e.g., four for nucleic acid synthesis. At these stations,multiple tube ends are carried along by the motor and lead screws, withall the tube ends pointing to the center of the well.

[0060] For DNA synthesis, the two stations that can have multipledispensing are the capping stations (Cap A and Cap B need not bedispensed at the same exact moment, but are always used together) andthe amidites. For the amidites, one monomer usually is dispensed out ofthe four at each well. However, in creating combinatorial libraries, onecan deliver a mixture of amidites to each of the wells, e.g., 25% of allfour, 50% of each of the purines or pyrimidines, etc. Using multipleheads saves stations for other uses. Alternatively, four separatestations can be dedicated to delivering one of each of the bases.

[0061] To change the location of a particular dispensing process, oneremoves the dispensing station and places it at another station, e.g.,by screwing it in. Just like drain stations, moving, adding, or deletinga dispensing station is simple. Thus, changing the set of chemicaladditions to satisfy a different chemical synthesis protocol is easy,making this combinatorial chemistry synthesis machine extremelyflexible.

[0062] F. TEMPERATURE CONTROLLING SYSTEM AND OPTICAL ANALYZING SYSTEM

[0063] Other processes (such as heating or analysis) can be placed atany station in the cycle. The heating process, for example, will beachieved by, for example, mounting a heat lamp in location available forthe dispensing stepper motor/lead screw assembly (using the samemounting screw holes).

[0064] The optical analysis can be done by placing a fiber opticemitter/detector in the stepper motor/lead screw assembly in place of adispensing tube end. This will be stepped through the eight wells in thereaction mount just as the dispensing tip is. At each well, the detectorcould assess the status of the reaction. For example, by measuring colorat the deblocking step in a nucleic acid synthesis, stepwise couplingefficiency can be established for each well. Other processes could beenvisioned to be mounted at the stations in lieu of the dispensingmotors. Moving, adding, deleting these processes is as easy as adjustingdispensing. Again, this increases the flexibility of this machine,making it capable of a variety of chemical protocols.

[0065] G. CONTAINMENT SYSTEM

[0066] The chemical protocol preferably takes place in an inertenvironment. Thus the airspace in which the process occurs (that aroundthe rotating carousel) can be isolated in a containment chamber. In oneembodiment, the containment is divided into two parts, an upper chamberand a lower chamber. The upper chamber is enclosed by a Plexiglaspolygonal tube. Inside is the motor that turns the reaction disc and thedispensing station. This is all above the reaction disc. The lowerchamber encloses the reaction disc itself, and the vacuum drain system,all inside a Plexiglas tube. The lower chamber is mounted on rails andis raised and lowered by a pneumatic cylinder. The lowered positionallows operator access to the disc (for cleaning and removal of theproduct). In the raised position, the lower chamber is pressed againstthe bottom of the upper chamber forming a seal (using closed cell foamas the gasket material). The disc mates with the main motor and is inposition for synthesis.

[0067] A non-reactive gas, such as nitrogen or argon, is pumped into thechamber from above and a regulator controls the inflow. A small outletvalve can be located on the bottom of the lower half of the chamber.This provides a constant flow of inert gas from above to below, pushingany chemical fumes away from the reaction wells. In addition, since allthe electronics (motors) are located above the reaction disc, thisensures that no flammable fumes can diffuse up to where the motors are.

[0068] The majority of the inert gas that flows into the chamber leavesthrough the drain system. Since the drain is not continuously activated(but instead at every “step”), there is a discontinuous requirement forinert gas in the chamber. When the vacuum valve opens (e.g., every 15seconds in a 24-station apparatus during nucleotide synthesis), it alsowill pull in a large volume of inert gas. To feed this gas flow throughthe vacuum system, and to avoid large pressure differences inside thechamber, a bellows system is attached to the chamber. When the drain isactivated, the large volume of inert gas needed comes from the bellows,which contract. The inert gas inflow through the regulator is set suchthat when the drain is not active, the bellows expand to full size. Anemergency relief valve is located on the bellows to avoid overpressure.

[0069] Since the efficiency of the coupling step of the synthesis ispartly a function of temperature, the inert gas that flows into thechamber is preheated. This is done with a gas heater controlled by a PIDcontroller along with a Drierite™ (WA Hammond Drierite) container thatserves as a heat reservoir.

[0070] H. PROGRAMMABLE COMPUTER

[0071] The apparatus also includes a programmable computer that controlsthe activity of all the systems. For example, a personal computerrunning an Intel Pentium processor running Windows 3.1 can control theentire machine. The control software can be written in C. The syringepumps are controlled via RS-232 bus (serial port), all the valves forpneumatic and keeping the reagents stored under inert gas are controlledby a digital I/O board with its signals run through solid state relays.Motor control is done through motion controller cards.

[0072] Polymer synthesis, for example, can involve the addition ofdifferent monomer units to nascent polymers in each of the wells. Inthis way a library is created. The computer can be programmed to producepolymers of different specific sequences in each of the wells. Thisinvolves programming the computer to add a specific monomer to each wellduring each rotation. Thus, the computer must know what reaction mountis docked at a fluid delivery station that delivers a monomer, whatrotation cycle is being carried out, and which delivery tube deliverswhich monomer.

[0073] III. PROCESSES FOR CONCURRENT CHEMICAL SYNTHESIS

[0074] This invention also provides a method for performing in parallela series of physical steps in a chemical reaction protocol, wherein theprotocol generates a chemical linkage on a parent molecule. The protocolcan result in a linkage between a parent polymer and a monomer unit,such as a nucleotide in the production of nucleic acids, an amino acidin the production of polypeptides, a monosaccharide in the production ofpolysaccharides and analogs thereof. It also can result in thealteration of a parent scaffold molecule. For example, the alterationcan be the generation of a new chemical linkage between the parentmolecule and an exogenous component. The alteration also can be themodification of an existing linkage in the parent molecule, e.g.,oxidation or reduction of existing bonds. Because the apparatus of thisinvention can be programmed to generate specific linkages in every well,the apparatus is well suited to the creation of libraries of compounds.

[0075] The method involves rotating the carousel step-wise for at leastone fill turn, and docking the reaction mounts at a station at eachstep. With each incremental step, a physical step in the protocol isperformed on at least one well (preferably all the wells) of a pluralityof reaction mounts (preferably all the reaction mounts) docked at thestation. A rotation of the carousel results in the full series ofphysical steps being performed in each of the reaction wells. Thus, withevery rotation of the carousel in which every station is in action, anew linkage is generated in the parent molecule attached to the solidsupport of a well. In this way, a polymer chain can be extended by thedesired number of monomer units by rotating the carousel one time foreach unit to be added.

[0076] For the following reasons, in performing the method of thisinvention, one will rotate the carousel through an initial rotation anda final rotation in order to initialize the wells and complete a cycleof the protocol in each well. Initialization is required in order tohave the reaction stage of the molecules in every mount correspond tothe station at which the mounts are docked. Completion is required inorder to ensure that all the steps of the protocol are performed on themolecules of every reaction well.

[0077] The stations are arranged around the carousel in a sequence fromthe first station dedicated to performing a first step, to a laststation dedicated to performing the last step. Because the stations arearranged in a circle, the last station is adjacent to the first station.Before the initial rotation, the parent molecules in all of the reactionwells are in the same state, prepared for the first step in thereaction. During the first rotation, the stations at which a reactionmount docks before the first station remain dormant during the docking:The first physical step is performed on a reaction mount only upondocking of the mount with the first station. Therefore, after the firstcomplete rotation of the carousel, only first reaction mount, i.e., themount initially docked at the first station, has had the entire processcarried out on it. The second reaction mount has had all but the laststep carried out, the third reaction mount has had all but the last twosteps carried out, etc., until the last mount, which began at the secondstation, has had no steps performed on it. Thus, at the end of the firstcycle, all the reaction mounts are initialized, with each mount preparedto have the next step carried out at the next station, but only thefirst mount having completed a cycle.

[0078] In order to complete each step of the protocol on the reactionmounts at some intermediate step of the reaction, the carousel must berotated a final time. In the final rotation, no steps are carried out onthe first reaction mount, which has already completed the cycle. Onlythe last step is performed on the second reaction mount. Only the lasttwo steps are performed on the second reaction mount. All the steps areperformed on the last reaction mount. Thus, by performing an initial andfinal cycle, each reaction mount has had all the physical stepsperformed on it once.

[0079] After the first cycle, the carousel can be rotated any number ofintermediate cycles to add a new linkage to each of the reaction mountsduring each cycle.

[0080] The practitioner may desire to create compounds with differentnumbers of linkages. For example, one may wish to prepare certainmembers of a nucleic acid library with 50 nucleotides and others with 40nucleotides. The apparatus of this invention can accomplish this withproper programming. The method involves allowing a particular reactionwell to sit out one or more intermediate cycles so that a linkage orlinkages are not created in that particular parent molecule. Thus,entire reaction mounts or particular wells of a reaction mount can sitout any cycle.

[0081] Any chemical reaction protocol can be broken down into a numberof physical steps by which the process is carried out. These stepsinclude adding a liquid to a well, removing liquid from a well andwaiting (incubate for a time, or a null step). Adding liquid to a welland removing it from the well can be combined at a single station tocreate a wash step. By properly arranging stations to perform thesesteps, and by adding appropriate liquids at appropriate steps, thereaction protocol can be carried out.

[0082] For example, phosphoramidite chemistry results in the creation ofa phosphotriester linkage between a parent nucleic acid and anucleotide, thereby adding a nucleotide to a parent polynucleotidechain. (The phosphodiester linkage is ultimately generated by removingprotecting groups from the phosphate, generating a hydroxyl linkage.) Aphosphoramidite chemistry protocol generally involves the followingreaction steps: Deprotection of a protected sugar moiety on a parentnucleic acid; coupling of a protected nucleotide to the deprotectedmoiety to create a phosphite triester linkage; oxidation of thephosphite triester linkage to generate a phosphotriester linkage; andcapping of unreacted nucleic acids. These reaction steps can, in turn,be dissembled into a number of physical steps. Stations can be arrangedaround the carousel to carry out these physical steps. In the case of anapparatus with 24 stations, certain of the stations can be dedicated toincubation or to washing a support more than once before proceeding tothe next step. A washing station can be created by attaching a fluiddelivery module at the station and attaching a vacuum line to theconduit connector at that station. In this example, the fluid deliverysystem includes several stations. Each station is connected to a supplyof the appropriate liquid to be dispensed to the well.

[0083] Typically, polymers are built by the iterative addition ofdifferent monomers. For example, a DNA molecule will be comprised of thefour bases, A, T, G and C (or other modified bases, such as I).Polypeptides will be comprised of the 20 naturally occurring aminoacids, or other non-naturally occurring analogs. Thus, the fluiddispenser at a monomer dispensing station will include as many tubes asthere are monomers. In the case of nucleic acids, this generally meansfour tubes, each of which dispenses one of the nucleotides.Alternatively, there could be four sequential stations, each dedicatedto delivering one of the bases. In this arrangement, the other stationsact as incubation stations when the particular nucleotide is not to bedelivered to a well of the reaction mount. In the case of polypeptides,it may be necessary to dedicate several stations in sequence to theaddition of various sub-sets of the 20 amino acids. In this case,certain of the stations will act as null stations if the particularamino acid is not delivered at that station. In preparing a library ofnucleic acid or polypeptide molecules, the programmable computer will beprogrammed with the identity of all the sequences to be prepared. Thecomputer will dispense the appropriate monomer to the appropriate wellat the appropriate cycle.

[0084] The arrangement of stations to perform other chemical protocolsis discussed in the Examples and will be apparent to one skilled in theart of solid phase chemical synthesis.

EXAMPLE

[0085] The following examples are presented by way of illustration, notlimitation.

[0086] A. INTRODUCTION

[0087] All chemical protocols are divided into reaction steps which, inturn, are divided into the physical steps. The physical steps includedelivering fluid to a well, draining fluid from a well, washing a welland incubating (wait or null step). Washing involves both adding fluidto a well and draining the fluid from the well. This can be performed intwo sequential stations dedicated to fluid delivery and fluid draining,or by combining fluid delivery and fluid draining at the same station.More specifically, a fluid delivery module can be set up at the stationand a vacuum line can be connected to engagement ports at the station.Incubation steps also can be adapted for heating, or analysis of theliquid (e.g., calorimetric). For example, a reaction step could involveadding liquid comprising a reactant to a well, incubating the well for aspecified period of time defined by one or more incremental steps,draining the liquid from the well, and washing the well at least once.

[0088] Once all the reaction steps in the protocol are divided intophysical steps, the number of steps is adjusted to the number ofstations in the apparatus, e.g., 24, by the addition of appropriate waitsteps or breaking a wash step into two steps (deliver liquid/drainliquid), for example. Then, the fluid delivery modules and vacuum linesare attached to the appropriate stations to carry out the physical stepsin sequence. Then, the computer can be programmed to deliver theappropriate reactants to designated wells in designated cycles, so as toproduce the desired chemical library. Also, the practitioner can addtemperature control or liquid analysis steps as desired.

[0089] In one embodiment, the chemical library is a library of polymers.Presented here are chemical protocols and physical steps that one canperform on an apparatus of this invention to carry out the protocol.

[0090] B. DNA—PHOSPHITE TRIESTER APPROACH

[0091] DNA synthesis generally involves the steps of deprotection,condensation, oxidation and capping. In the phosphite-triester method,deprotection involves removing an acid-labile DMTr group from the 5′-OHof a sugar moiety. The condensation step involves coupling excessactivated monomer to the growing chain. The oxidation step involvesoxidation of 3′-5′ internucleotide phosphite triester linkage to a morestable phosphotriester linkage. (After completion of the process, thepolymers are treated to remove protecting groups, thereby generating thephophodiester linkages.) The capping step involves capping 5′-hydroxylgroups that failed to condense as acetate esters. This step is optionaldepending on how the cycle is optimized. When more than enough time isallowed for all of the reactions to proceed to completion, this step canbe omitted. The general protocol can involve the following reactionsteps:

[0092] 1. washing the support;

[0093] 2. dispensing a liquid comprising a deblocking agent to removethe protecting group;

[0094] 3. draining the liquid comprising the deblocking agent;

[0095] 4. washing the support;

[0096] 5. dispensing a liquid comprising a coupling activator;

[0097] 6. dispensing a liquid comprising a protected nucleotide;

[0098] 7. draining the liquid comprising a protected nucleotide;

[0099] 8. dispensing a liquid comprising a capping agent;

[0100] 9. draining the liquid comprising the capping agent;

[0101] 10. washing the support;

[0102] 11. dispensing a liquid comprising an oxidizer; and

[0103] 12. draining the liquid comprising the oxidizer.

[0104] Repeat steps 1-12 until nucleotide sequence complete

[0105] These reaction steps can be performed in the following physicalsteps. An apparatus of this invention was set up having 24 stations inthe following arrangement. Each cycle of the carousel resulted in theaddition of one nucleotide linkage to the parent nucleotide. Thephysical step indicates the system (fluid delivery/drain/null) to bededicated to each station. Station: Action: Physical step: 1Acetonitrile wash 80 μL dispensed and drained 2 Acetonitrile wash 80 μLdispensed and drained 3 TCA deblock 40 μL dispensed 4 Drain Drain 5 TCAdeblock 40 μL dispensed 6 Drain Drain 7 Acetonitrile wash 80 μLdispensed and drained 8 Acetonitrile wash 80 μL dispensed and drained 9Tetrazole Activator 10 μL dispensed 10 Amidite 10 μL of A, T, C, or Gdispensed 11 5′ Special 10 μL of AminoLink (or Null) 12 Incubate Null 13Incubate Null 14 Incubate Null 15 Incubate Null 16 Incubate Null 17Drain Drain 18 Acetonitrile wash 80 μL dispensed and drained 19 Caps Aand B 12 μL of each Cap dispensed 20 Incubate Null 21 Drain Drain 22Acetonitrile wash 80 μL dispensed and drained 23 Oxidizer 12 μLdispensed 24 Drain Drain

[0106] The above sequence is for synthesis of oligonucleotides. Otherconfigurations are possible for other types of polymer synthesis. Thestep time in this example was 15 seconds, thus a full cycle of the discrequires about 6 minutes.

[0107] For synthesis of 50 mer oligos, the first 50 cycles involve thedispensing of one of the four amidites at each well throughout thesynthesis. The 5 prime AminoLink is not used during these cycles,effectively being an incubation station. After the 50 mers arecompleted, another cycle can be done to attach a 5 prime aminolink. Inthis case, the standard amidites are used. In one particular protocol,the step time is increased to 20 seconds for the aminolink cycle toaccommodate longer coupling time. Note that other monomers could beincluded in this system. They could be attached along with the standardamidites, or with a unique cycle if they require different timing.

[0108] The Teflon frits which serve as the substrate are round, 0.240 mm(just under 0.001″) in thickness, and {fraction (7/32)} inches indiameter. They are die cut from PerSeptive™ MemSyn sheets.

[0109] C. DNA—PHOSPHOTRIESTER APPROACH

[0110] In the phosphotriester approach to DNA synthesis, deprotectioninvolves removing the acid-labile dimethoxytrityl (DMTr) or Pixyl (Px)group from the 5′ hydroxyl (—OH). The condensation step involvescondensing the monomer (as its triethylammonium salt) with the free5′-hydroxyl group of the growing chain in the presence of a couplingagent and a catalyst. The oxidation step is not required since thephosphate moiety is already in the more stable form. The capping step isnot necessary because there is an insignificant amount of unreacted 5′hydroxyl groups using the phosphotriester method. Note that for thewashes in between the reaction steps, the phosphite-triester method usesacetonitrile (ACN), whereas the phosphotriester method uses pyridine anddichloroethane.

[0111] D. RNA

[0112] RNA synthesis is similar to DNA synthesis: it involves step-wiseaddition of a protected ribonucleoside phosphoramidite to the growingRNA chain that is attached to a solid support. For RNA synthesis,coupling takes much longer (˜10 min) due to the extra protecting groupon the 2′-OH of the ribose sugar. Therefore, additional incubation stepsmust be factored into the station arrangement. Otherwise, all of thesteps in DNA and RNA syntheses—deprotection, condensation, oxidation,capping—are the same, employing the same reagents (except for themonomers and support-bound nucleosides). Therefore, the protocol for DNAsynthesis can be followed to make RNA.

[0113] E. DNA AND RNA ANALOGUES

[0114] 1. DNA/RNA hybrids

[0115] The practitioner can make DNA and RNA hybrids on the sameinstrument by supplying all of the appropriate monomers (A, T, C, G forDNA; A, U, C, G for RNA) and changing the coupling times depending onwhether one is adding a deoxyribonucleoside or a ribonucleoside.

[0116] 2. Backbone-modified nucleic acids

[0117] Backbone modifications to both DNA and RNA can be performed aswell. Phosphorothioate oligonucleotides can be made simply bysubstituting the oxidizing reagent with a sulfurizing reagent (slightlyincreased delivery volume but same reaction timing). The cyclefollows: 1) deprotection, 2) condensation, 3) sulfurization (with TETD,tetraethylthiuran disulfide in ACN), and 4) capping, with appropriatedraining and washing in between each of the four steps.

[0118] Another method for backbone modifications uses H-phosphonatechemistry. The protocol can include the following steps:

[0119] 1. Wash

[0120] 2. Deblock (at least once)

[0121] 3. Drain

[0122] 4. Wash (at least once)

[0123] 5. Couple (with 10 mM nucleoside H-phosphonate and activator)

[0124] 6. Drain

[0125] 7. Repeat steps 1-6 until nucleotide sequence complete*

[0126] 8. Final deblock of entire nucleic acid (at least once)

[0127] 9. Drain

[0128] 10. Wash (at least once)

[0129] 11. Oxidize ALL H-phosphonate bonds to phosphodiester linkages**

[0130] 12. Drain

[0131] 13. Wash (at least once).

[0132] *Capping is omitted since activator (pivaloyl chloride) also actsas capping reagent.

[0133] ** Instead of oxidation with iodine, use alternative reagent toconvert H-phosphonate bonds to phosphorothioates, phosphoramidites orphosphotriesters.

[0134] F. POLYPEPTIDES

[0135] The addition of amino acids onto a nascent polypeptide chaininvolves three chemical reactions: 1. Deprotection—protecting group(Fmoc or Boc) removed to make alpha-amino group on end of growingpeptide chain available. 2. Coupling—amino acid residue is firstactivated into an active ester and then forms an amide bond with thedeprotected alpha-amino group on end of growing peptide chain. 3.Capping [optional]—unreacted alpha-amino groups are capped with the samereagent used in DNA/RNA synthesis.

[0136] Two protocols exist for peptide synthesis, each named after theprotection strategy. In Fmoc synthesis, the base-labile protecting group(Fmoc) is removed at each cycle. At the end of the synthesis, the sidechain protecting groups are removed by a weak acid, which also cleavesthe bond anchoring the peptide to the support. In Boc chemistry, the Bocprotecting group is acid-labile and can be removed with a mild acid. Astrong acid is used for the final deprotection and cleavage step. Fmocchemistry is preferred due to the milder conditions (less causticreagents).

[0137] A protocol for peptide synthesis using Fmoc chemistry can includethe following steps:

[0138] 1. Deprotect (at least once)—piperidine, 2×-7 minutes

[0139] 2. Drain

[0140] 3. Wash (at least once)—N-methylpyrrolidone (NMP) ordimethylformamide (DMF), 6×

[0141] 4. Couple—18 seconds activation+35 minutes coupling

[0142] 5. Drain

[0143] 6. Cap [optional]—1 minute

[0144] 7. Drain

[0145] 8. Wash (at least once)—NMP or DMF, 3×.

[0146] Repeat steps 1-8 until amino acid sequence is complete

[0147] A protocol for peptide synthesis using Boc chemistry can includethe following steps:

[0148] 1. Wash (at least once)—dichloromethane (DCM), 1×

[0149] 2. Deprotect (at least once)—trifluoroacetic acid (TEA), 2×-6minutes

[0150] 3. Drain

[0151] 4. Wash (at least once)—dichloromethane (DCM), 1×

[0152] 5. Wash (at least once)—NMP or DMF, 6×

[0153] 6. Couple—18 seconds activation+35 minutes coupling

[0154] 7. Drain

[0155] 8. Cap [optional]—1 minute

[0156] 9. Drain

[0157] 10. Wash (at least once)—NMP or DMF, 3×.

[0158] Repeat steps 1-10 until amino acid sequence is complete

[0159] G. PEPTIDE NUCLEIC ACIDS (PNA'S)

[0160] PNA oligomer synthesis uses a modified solid-phase peptidesynthesis protocol, similar to the Boc cycle above. Instead of couplingamino acid residues as in peptide synthesis, PNA monomers (A, C, G, T)containing the BOC protecting group are activated with a uronium saltactivator. During synthesis, it is possible to modify the PNA oligomerwith amino acids and various labels such as fluorescein, biotin orrhodamine.

[0161] A protocol for PNA synthesis using modified Boc chemistry caninvolve the following steps:

[0162] 1. Wash (at least once)—dichloromethane (DCM), 1×

[0163] 2. Deprotect (at least once)—trifluoroacetic acid (TFA), 2×-6minutes

[0164] 3. Drain

[0165] 4. Wash (at least once)—dichloromethane (DCM), 3×

[0166] 5. Wash (at least once)—NMP or DMF, 6×

[0167] 6. Couple—1 minute activation+35 minutes coupling

[0168] 7. Drain

[0169] 8. Cap—1 minute

[0170] 9. Drain

[0171] 10. Wash (at least once)—NMP or DMF, 3×.

[0172] Repeat steps 1-10 until PNA sequence is complete

[0173] The present invention provides novel materials and methods forconcurrent chemical synthesis. While specific examples have beenprovided, the above description is illustrative and not restrictive.Many variations of the invention will become apparent to those skilledin the art upon review of this specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

[0174] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument Applicants do not admit that any particular reference is “priorart” to their invention.

What is claimed is:
 1. An apparatus comprising: a) a carousel that isrotatable around an axis, the carousel comprising a plurality ofreaction mounts, each reaction mount comprising at least one reactionwell arranged on a radius with respect to the axis, the radii spacedapart at equal angles, whereby the wells are arranged in at least oneconcentric circle around the axis; b) a rotator that rotates thecarousel step-wise around the axis, each incremental step docking eachof the reaction mounts at a separate station; c) a fluid delivery systemthat delivers liquid to at least one reaction well in each of aplurality of docked reaction mounts; d) a drain system that drainsliquid by differential pressure from at least one reaction well of eachof a plurality of docked reaction mounts; and e) a programmable digitalcomputer that controls the rotator, the fluid delivery system and thedrain system.
 2. The apparatus of claim 1 wherein: (i) each reactionwell comprises a drainage hole; (ii) the carousel comprises a platewhich comprises a plurality of liquid conduits that connect with thedrainage holes and are engagable with the drain system; and (iii) thedrain system is a vacuum drain system comprising: (1) a plurality ofvacuum lines that connect with vacuum source and (2) conduit engagementmeans that engage the vacuum lines with a plurality of the liquidconduits when the reaction mounts are docked at a station, wherebyliquid in the reaction wells is drained through the vacuum lines.
 3. Theapparatus of claim 2 wherein: (i) each liquid conduit comprises: (1) adepression in the plate below the reaction mount which forms a chamberwith the reaction mount, wherein the chamber communicates with thedrainage holes of the reaction mount; (2) an exit port exiting under theplate; and (3) a bore through the plate the connects the chamber withthe exit port; and (ii) the conduit engagement means comprises: (1) anon-rotating connector plate positioned under the carousel; theconnector plate having an engagement port that is engagable with theexit port positioned at each station, wherein each of a plurality of theengagement ports is connected to a vacuum line; and (2) an actuator thatraises the connector plate to engage the plurality of engagement portswith the plurality of exit ports.
 4. The apparatus of claim 2 whereinthe fluid delivery system comprises: (i) an assembly positioned abovethe carousel, the assembly comprising a plurality of dispensing modulesmounted at each of a plurality of the stations, each dispensing modulecomprising a dispensing head adapted to deliver fluid to the well of areaction mount docked at the station; (ii) a plurality of fluiddispensers, each dispenser adapted to dispense an amount of fluid; (iii)a plurality of fluid lines, each fluid line connecting a fluid dispenserto a dispensing head.
 5. The apparatus of claim 2 wherein the number ofreaction mounts equals the number of stations.
 6. The apparatus of claim2 wherein the carousel comprises 24 reaction mounts.
 7. The apparatus ofclaim 2 wherein the reaction mounts each comprise 8 reaction wells. 8.The apparatus of claim 2 wherein the fluid delivery system deliversliquid to at least one reaction well in each of at least 3 dockedreaction mounts and the vacuum drain system drains liquid from at leastone reaction well of each of at least 3 docked reaction mounts.
 9. Theapparatus of claim 2 further comprising a temperature controlling systemthat regulates the temperature of at least one reaction mount docked ata station.
 10. The apparatus of claim 2 further comprising an opticalanalyzing system that optically analyzes fluid in a well of at least onereaction mount docked at a station.
 11. The apparatus of claim 4wherein: (i) each reaction mount comprises a plurality of wells; (ii)each dispensing module comprises a motor that moves the dispensing headto positions suitable for delivering fluid to each of the plurality ofwells.
 12. The apparatus of claim 4 wherein at least one stationcomprises both a dispensing module and an engagement port connected to avacuum line.
 13. The apparatus of claim 4 wherein each reaction mountcomprises a plurality of wells; the wells being spaced apart about thedistance of wells in a row of a 96-well microtiter plate.
 14. Theapparatus of claim 4 further comprising an airtight chamber thatcomprises the rotator, the dispensing assembly, the carousel and theconnector plate.
 15. The apparatus of claim 11 wherein at least onedispensing head is connected to a plurality of fluid dispensers by fluidlines.
 16. The apparatus of claim 14 wherein the chamber comprises anupper chamber and a lower chamber wherein the upper chamber comprisesthe rotator and the dispensing assembly, and the lower chamber comprisesthe carousel and the connector plate, and wherein the lower chamber canbe in a raised or lowered position with respect to the upper chamber,and wherein in the raised position, the chamber forms an airtight seal.17. The apparatus of claim 14 comprising a regulator which regulates adirectional flow of a gas to the upper chamber.
 18. The apparatus ofclaim 16 further comprising a bellows connected to the regulator and tothe upper chamber which functions as a reservoir for the gas.
 19. Amethod for performing in parallel a series of physical steps in achemical reaction protocol, wherein the protocol generates a chemicallinkage in a parent molecule, the method comprising: a) providing acarousel that is rotatable around an axis, the carousel comprising aplurality of reaction mounts, each reaction mount comprising at leastone reaction well arranged on a radius with respect to the axis, theradii spaced apart at equal angles, whereby the wells are arranged in atleast one concentric circle around the axis, wherein each well comprisesthe parent molecule attached to a solid support; b) rotating thecarousel step-wise around the axis at least once, each incremental stepdocking each of the reaction mounts at a separate station, wherein (1)each station is dedicated to perform a physical step in the seriesduring a docking, wherein the physical steps include adding a liquid toa well, draining a liquid from a well, and incubating; and (2) thestations are arranged to perform the series of physical steps insequence; and c) performing, with each rotation of the carousel, theseries of physical steps in a reaction well of each of at least two ofthe reaction mounts, whereby a chemical linkage is generated in theparent molecule.
 20. The method of claim 19 comprising rotating thecarousel a plurality of times.
 21. The method of claim 19 comprising,with at least one rotation of the carousel, performing the series ofsteps in a reaction well of all of the reaction mounts.
 22. The methodof claim 19 wherein the series of steps is not performed on a reactionwell of at least one reaction mount during at least one rotation,whereby the reaction mount skips the protocol during that rotation. 23.The method of claim 19 wherein the parent molecule is cleavable from thesolid support.
 24. The method of claim 19 wherein there are 24 stations.25. The method of claim 19 wherein the chemical linkage links acomponent to the parent molecule.
 26. The method of claim 19 carried outin an inert atmosphere.
 27. The method of claim 19 wherein the physicalsteps further include washing a well, wherein washing comprises bothadding fluid to a well and draining fluid from a well at a singlestation.
 28. The method of claim 19 wherein the steps include heating awell.
 29. The method of claim 19 wherein the steps include opticallyanalyzing a well.
 30. The method of claim 19 wherein the chemicallinkage is selected from at least one of a phosphodiester bond, aphosphorothioate bond, a phosphonate bond, a phosphoramidate bond, anamide bond, an imine bond, a carbamate bond, an azo bond, a sulfonebond, a sulfonide bond, a sulfonamide bond, a sulfide bond, a disulfidebond, an ether bond, an ester bond, a thiourea bond, a urea bond and acarbon-carbon bond.
 31. The method of claim 19 wherein the chemicallinkage generates a new chemical linkage in the parent molecule but doesnot link a component to the parent molecule.
 32. The method of claim 25wherein the parent molecule is a polymer and the component is a monomer.33. The method of claim 25 wherein the parent molecule is a scaffoldmolecule and the component is an atom or molecule.
 34. The method ofclaim 25 wherein a different fluid comprising a different component isadded to different wells, wherein the different fluid added to a well iscontrolled by a programmable computer, whereby a library of differentparent molecules is created.
 35. The method of claim 32 wherein thepolymer is a nucleic acid.
 36. The method of claim 32 wherein thepolymer is DNA.
 37. The method of claim 32 wherein the polymer is RNA.38. The method of claim 32 wherein the polymer is a peptide nucleicacid.
 39. The method of claim 32 wherein the polymer is a polypeptide.40. The method of claim 34 wherein the computer directs the generationof a library of polymers of predetermined sequence.
 41. The method ofclaim 35 wherein the nucleic acid is coupled to a solid support in thewell and the series of physical steps includes, in sequence: (i) washingthe support; (ii) dispensing a liquid comprising a deblocking agent toremove the protecting group; (iii) draining the liquid comprising thedeblocking agent; (iv) washing the support; (v) dispensing a liquidcomprising a coupling activator; (vi) dispensing a liquid comprising aprotected nucleotide; (vii) draining the liquid comprising a protectednucleotide; (viii) dispensing a liquid comprising a capping agent; (ix)draining the liquid comprising the capping agent; (x) washing thesupport; (xi) dispensing a liquid comprising an oxidizer; and (xii)draining the liquid comprising the oxidizer.
 42. The method of claim 35wherein the monomer is a modified nucleotide comprising a minor groovebinder.
 43. The method of claim 35 comprising rotating the carousel toproduce a nucleic acid having between 5 and 200 nucleotides.
 44. Themethod of claim 39 comprising rotating the carousel to produce apolypeptide having between 5 and 50 amino acids.
 45. A method forperforming in parallel a series of physical steps in a chemicalprotocol, the method comprising the steps of: a) providing a carouselthat is rotatable around an axis, the carousel comprising a plurality ofreaction mounts, each reaction mount comprising at least one reactionwell arranged on a radius with respect to the axis, the radii spacedapart at equal angles, whereby the wells are arranged in at least oneconcentric circle around the axis, wherein each well comprises theparent molecule attached to a solid support; b) providing a rotator thatrotate the carousel step-wise around the axis, each incremental stepdocking the reaction mounts a station, wherein: (1) each station isdedicated to perform a physical step in the series during a docking and(2) the stations are arranged in series from an initial station thatperforms an initial physical step in a series of physical steps in achemical protocol to a final station that performs a final physical stepin the series of physical steps; c) performing an initial rotation ofthe carousel around the axis, wherein the stations begin to perform theseries of physical steps as a reaction mount docks at the initialstation; and d) performing a final rotation of the carousel around theaxis, wherein the stations cease to perform the series of physical stepsas a reaction mount docks at the final station; whereby the initial andfinal rotations result in one complete series of steps on a reactionwell of each reaction mount.
 46. The method of claim 45 furthercomprising performing at least one intermediate rotation between theinitial and final rotations.